Absolute configuration of aflastatin A, a specific inhibitor of aflatoxin production by Aspergillus parasiticus

Article abstract of DOI:10.1021/jo991284c

Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It has the novel structure of a tetramic acid derivative with a long alkyl side chain. The absolute configurations of 29 chiral centers contained in I were chemically elucidated in this study. First, four small fragment molecules were prepared from I or its methyl ether (2), and their absolute structures were assigned as N-methyl-D-alanine, (2S,4R)-2,4-dimethyl-1,6-hexanediol dibenzoate, (R)-3-hydroxydodecanoic acid, and (R)-1,2,4-butanetriol tribenzoate. Next, an acyclic fragment molecule 3 with 13 chiral centers was obtained from I by NaIO₄ oxidation, and its relative stereochemistry was elucidated by J-based configuration analysis. By analyzing coupling constants of ³J(H,H) and ^2,3J(C,H) and ROE data, the relative configuration of 3 was verified. Finally, by further J-based configuration analysis using a fragment molecule 7 prepared from 2 with 28 chiral carbons, all relative configurations in the alkyl side chain of I were clarified. By connecting these relative configurations with the absolute configurations of first four fragment molecules, the absolute stereochemistry of 1 was fully determined.

Full text of DOI:10.1021/jo991284c

438
J . Org. Chem. 2000, 65, 438-444
Absolu te Con figu r a tion of Afla sta tin A, a Sp ecific In h ibitor of
Afla toxin P r od u ction by Asper gillu s pa r a siticu s
Hiroyuki Ikeda,1a Nobuaki Matsumori,1b,e Makoto Ono,1c Akinori Suzuki,1a Akira Isogai,1d
Hiromichi Nagasawa,1a and Shohei Sakuda*,1a
Department of Applied Biological Chemistry and Biotechnology, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, J apan, Research Institute, Morinaga and Co. Ltd., Tsurumi-ku,
Yokohama 230-8504, J apan, and Graduate School of Biological Science, Nara Institute of Science and
Technology, Ikoma, Nara 630-0101, J apan
Received August 11, 1999
Aflastatin A (1) is a specific inhibitor of aflatoxin production by Aspergillus parasiticus. It has the
novel structure of a tetramic acid derivative with a long alkyl side chain. The absolute configurations
of 29 chiral centers contained in 1 were chemically elucidated in this study. First, four small
fragment molecules were prepared from 1 or its methyl ether (2), and their absolute structures
were assigned as N-methyl-D-alanine, (2S,4R)-2,4-dimethyl-1,6-hexanediol dibenzoate, (R)-3-
hydroxydodecanoic acid, and (R)-1,2,4-butanetriol tribenzoate. Next, an acyclic fragment molecule
3 with 13 chiral centers was obtained from 1 by NaIO4 oxidation, and its relative stereochemistry
3
was elucidated by J -based configuration analysis. By analyzing coupling constants of J H,H and
2,3
J
and ROE data, the relative configuration of 3 was verified. Finally, by further J -based
C,H
configuration analysis using a fragment molecule 7 prepared from 2 with 28 chiral carbons, all
relative configurations in the alkyl side chain of 1 were clarified. By connecting these relative
configurations with the absolute configurations of first four fragment molecules, the absolute
stereochemistry of 1 was fully determined.
Aflatoxins belong to a group of mycotoxins produced
statin A inhibits aflatoxin production by Aspergillus
parasiticus at low concentrations without essentially
affecting the growth of A. parasiticus. We have reported
the planar structure of 1.5b,c It is a novel tetramic acid
derivative with a long alkyl side chain. The side chain is
polyhydroxylated and acyclic except for a tetrahydropy-
ran ring moiety. There are 29 chiral centers in 1.
Determination of the absolute configuration of 1 is very
important for further studies such as chemical synthesis,
structure-activity relationship, and its mode of action.
However, there has been no information regarding the
stereochemistry of 1 except for a relative stereochemistry
around the tetrahydropyran ring. In this paper, we
describe the elucidation of the absolute configuration of
1.
Since crystals of 1 or its derivative suitable for X-ray
analysis have not been obtained, we tried to determine
its absolute configuration chemically. It is known that
three fragment molecules, 3-5, were obtained from 1
(Figure 1).5c,6 These three and two other fragment
molecules, 6 and 7, were used for determination of the
absolute configuration of 1. Our strategy was as follows:
(1) absolute configurations at C5′, C4 and C6, C33, and
C39 of 1 should be clarified by determination of the
absolute structures of 4-6, (2) since 3 was acyclic and
all chiral carbons in 3 were present in a 1,2- or 1,3-
methine system, the relative configuration of 3 should
be determined by the J -based configuration analysis,7
which could afford the relative configurations from C10
by some strains of Aspergillus parasiticus, Aspergillus
flavus, Aspergillus nomius, and Aspergillus tamarii.
Since they have extremely potent carcinogenicity toward
mammals and are contaminants in a wide variety of food
commodities, they are generally recognized not only as
toxic contaminants in foods and feeds, but also as a
certain risk factor for liver cancer in humans.2 Therefore,
the control and management of aflatoxins have been
issues of concern.3
A specific inhibitor for aflatoxin biosynthesis may be
a good candidate for a useful drug to protect foods and
feeds from aflatoxin contamination. It is expected to
depress aflatoxin contamination without rapid spread of
drug-resistant strains since, unlike fungicides, it does not
kill the producer of aflatoxin. Until now, some natural
products and synthetic pesticides have been known to
have inhibitory activity toward aflatoxin production.4
However, none of them have been used in protecting
agricultural products from aflatoxin contamination. Re-
cently, during the course of our screening, aflastatin A
(1) was isolated from the mycelia of Streptomyces sp.
MRI142 as an inhibitor of aflatoxin production.5a Afla-
* To whom correspondence should be addressed. E-mail: asakuda@
mail.ecc.u-tokyo.ac.jp FAX: +81-3-5841-8022
(1) (a) Department of Applied Biological Chemistry, The University
of Tokyo. (b) Department of Biotechnology, The University of Tokyo.
(c) Morinaga and Co., Ltd. (d) Nara Institute of Science and Technology.
(e) Present address: Department of Chemistry, Osaka University, 1-16
Machikaneyama, Toyonaka, Osaka 560-0043, J apan.
(2) Massey, T. E.; Stewart, R. K.; Daniels, J . M.; Liu, L. Proc. Exp.
Biol. Med. 1995, 208, 213-227.
(5) (a) Ono, M.; Sakuda, S.; Suzuki, A.; Isogai, A. J . Antibiot. 1997,
50, 111-118. (b) Sakuda, S.; Ono, M.; Furihata, K.; Nakayama, J .;
Suzuki, A.; Isogai, A. J . Am. Chem. Soc. 1996, 118, 7855-7856. (c)
Ono, M.; Sakuda, S.; Ikeda, H.; Furihata, K.; Nakayama, J .; Suzuki,
A.; Isogai, A. J . Antibiot. 1998, 51, 1019-1029.
(6) Sakuda, S.; Ono, M.; Ikeda, H.; Inagaki, Y.; Nakayama, J .;
Suzuki, A.; Isogai, A. Tetrahedron Lett. 1997, 38, 7399-7402.
(3) Goldblatt, L. A. Aflatoxin; Academic Press: New York, 1969; pp
13-46.
(4) (a) Zaika, L. L.; Buchanan, R. L. J . Food Prot. 1987, 50, 691-
708. (b) Wheeler, M. F.; Bhatnagar, D. Pestic. Biochem. Physiol. 1995,
52, 109-115. (c) Wheeler, M. H.; Bhatnagar, D.; Rojas, M. G. Pestic.
Biochem. Physiol. 1989, 35, 315-320.
10.1021/jo991284c CCC: $19.00 © 2000 American Chemical Society
Published on Web 12/31/1999

Absolute Configuration of Aflastatin A
J . Org. Chem., Vol. 65, No. 2, 2000 439
F igu r e 1. Procedures for the degradation of aflastatin A (1). (a) NaIO4; NaBH4. (b) NaIO4; NaBH4; Ac2O, pyridine; NaOMe. (c)
NaIO4. (d) NaIO4; 3 N HCl. (e) O3; Me2S; LiAlH4; BzCN, tri-n-butylamine. (f) 5% HCl-MeOH. (g) NaIO4; NaBH4; 3 N HCl; BzCl,
pyridine. (h) O3; NaBH4; Ac2O, pyridine; NaOMe; Dowex-50W (H+).
to C25 of 1, (3) the remaining relative configurations from
C6 to C10 and from C25 to C33 of 1 should be clarified
by the determination of those of the counterparts in 7,
which could also be analyzed by the J -based method, and
(4) by connecting the absolute configurations at C6 and
C33 of 1 with the relative configurations from C6 to C33
and from C33 to C37 of 1, the complete absolute config-
uration of 1 should be determined.
Sch em e 1
First, 4 was degraded to afford N-methylalanine (8)
and 2,4-dimethyl-1,6-hexanediol dibenzoate (9). The ab-
solute configuration of 8 was determined as D by Marfey’s
method.8 To determine the absolute configuration of 9,
optically active (2S,4S)- and (2R,4S)-9 were prepared
from cycloheximide (10) according to Scheme 1. Alkaline
degradation of 10 afforded (2S,4R)- and (2R,4R)-2,4-
dimethylcyclohexanone (11).9 Compound (2S,4R)-11 was
acetoxylated and converted to lactone (2S,4S)-12. LiAlH4
reduction of this lactone, followed by benzoylation, af-
forded (2S,4S)-9. Similarly, (2R,4S)-9 was obtained from
(2R,4R)-11. The 1H NMR spectrum of natural 9 was
identical with that of (2R,4S)-9, indicating that natural
9 had a syn stereochemistry. Comparison of the CD
spectrum of natural 9 with that of (2R,4S)-9 showed that
they were enantiomers. Thus, the configuration of natu-
ral 9 was assigned as (2S,4R). From the configurations
of 8 and 9, the absolute configurations at C5′, C4, and
C6 of 1 were determined as shown in Figure 1.
(7) (a) Matsumori, N.; Kaneno, D.; Murata, M.; Nakamura, H.;
Tachibana, K. J . Org. Chem. 1999, 64, 866-876. In the J -based
method, the configuration of a 1,2- or 1,3-methine system is deduced
from the result of its conformation analysis with the J values and NOE
(ROE) data. In the case of a 1,3-methine system separated by a
methylene group, both conformations for the two methine-methylene
systems are analyzed, respectively. The following J values are used
for the J -based configuration analysis in general.
Next, 1 was converted to its methyl glycoside 2. This
was oxidized with NaIO4, followed by NaBH4 reduction,
acid hydrolysis, and benzoylation, to afford 1,2,4-butan-
etriol tribenzoate (6), in which the configuration at C33
of 1 was maintained. The absolute configuration of 6 was
assigned as R by comparison of its CD spectrum with
that of an authentic sample, which was prepared from a
commercially available (R)-1,2,4-butanetriol. Since the
relative configuration of the tetrahydropyran ring has
been verified, the absolute configurations from C33 to
3
3
2
a: J H,H. b: J C,H. c: J C,H. The figures in parentheses represent the
values of 1,2-dioxygenated systems.
This method has been applied to determination of the configurations
of some natural products such as maitotoxin,7b-d dysiherbaine,7e and
amphidinol 3.7f (b) Matsumori, N.; Nonomura, T.; Sasaki, M.; Murata,
M.; Tachibana, K.; Satake, M.; Yasumoto, T. Tetrahedron Lett. 1996,
37, 1269-1272. (c) Sasaki, M.; Matsumori, N.; Maruyama, T.; Nono-
mura, T.; Murata, M.; Tachibana, K.; Yasumoto, T. Angew. Chem., Int.
Ed. Engl. 1996, 35, 1672-1675. (d) Nonomura, T.; Sasaki, M.;
Matsumori, M.; Murata, M.; Tachibana, K.; Yasumoto, T. Angew.
Chem., Int. Ed. Engl. 1996, 35, 1675-1678. (e) Sakai, R.; Kamiya, H.;
Murata, M.; Shimamoto, K. J . Am. Chem. Soc. 1997, 119, 4112-4116.
(f) Murata, M.; Matsuoka, S.; Matsumori, N.; Paul, G. K.; Tachibana,
K. J . Am. Chem. Soc. 1999, 121, 870-871.
(8) Marffey, P. Carlsberg Res. Commun. 1984, 49, 591-596.
(9) Bernard, C. L. J . Am. Chem. Soc. 1962, 84, 239-244.

440 J . Org. Chem., Vol. 65, No. 2, 2000
Ikeda et al.
3
2,3
F igu r e 2. Configurations and conformation of 3 established on the basis of J H,H
,
J
, and key ROEs. Arrows, broken lines
C,H
2,3
with arrows, and bold curves show 3J H,H
,
J
, and ROE, respectively. C9-C10 portion is depicted with a zigzag-type conformation.
Spectra were measured in CD3OD or pyridine-d5: CD3OD (3:1). Measured in pyridine-d5:CD3OD (3:1).
C,H
a
C37 of 1 were assigned as shown in Figure 1 based on
the configuration at C33 determined. From the optical
rotation value of 5, its absolute configuration was as-
signed as R,10 indicating that the configuration of C39 of
1 was R.
To elucidate the relative configuration of 3 by the
3
2,3
J -based method, J H,H and
J
values were obtained
C,H
from E.COSY, HETLOC,11,7a and phase-sensitive
HMBC12,7a spectra. Two different solvents, CD3OD and
CD3OD-pyridine-d5 (3:1), were used for the NMR mea-
surement to overcome the signal overlappings. As shown
in Figure 2, dominant conformations for C2-C3, C3-
C4, C4-C5, C5-C6, C6-C7, C7-C9, C10-C11, C11-
C12, C12-C13, C13-C15, and C15-C17 of 3 were
unambiguously assigned from the typical anti or gauche
J values and ROEs,7a which lead to the establishment of
configuration as threo, erythro, erythro, threo, threo, syn,
erythro, threo, threo, syn, and syn, respectively. For the
remaining C9-C10 bond in 3, the medium value of
3J H-9,H-10 (5.5 Hz) was observed, indicating that an
alternating conformational change occurs around the
F igu r e 3. Rotamers for C9-C10 of 3 (a), C4-C5 of 7 (b),
C29-C30 of 7 (c), and C6-C7 and C27-C28 of 7 (d).
3
2
bond. From the J C23,H-9 (3.5 Hz) and J C9,H-10 (-5.6 Hz)
values, a pair of alternating conformers with the erythro
configuration (Figure 3a) was easily assigned for C9-
C10 according to the J -based method.7a Thus, the total
relative configuration of 3 was determined as shown in
Figure 2, which afforded relative configurations from C10
to C25 of 1.
This relative configuration of 3 was partly confirmed
by using the acetonide method.13 Pentaacetonide of 3 (13)
was prepared, and its 13C NMR spectrum was analyzed.
Figure 4 shows the δ value of each carbon involved in
the five acetonide moieties, which was assigned by
analysis of COSY, FG-HMQC, FG-HMBC, and NOESY
spectra of 13. Since all these values were typical for the
case of the 1,3-diol system with syn relationship, all
relative configurations for C5-C7, C9-C11, and C13-
C15 were assigned as syn, which were completely con-
sistent with those obtained by the J -based method
mentioned above.
F igu r e 4. Structure of 13 and 13C assignments of its acetonide
moieties. Bold-faced numbers indicate δC values in benzene-
d6.
Finally, to determine the remaining configurations
from C6 to C10 and from C25 to C33 of 1, a large
fragment molecule (7) was prepared from 2 by ozonolysis
and subsequent reactions as shown in Figure 1. The J
values and ROEs used for determination of the configu-
rations from C4 to C8 and from C23 to C31 of 7 are
summarized in Table 1. Each configuration for C7-C8,
C23-C25, C25-C26, C26-C27, and C28-C29 of 7 was
assigned as erythro, syn, threo, threo, and threo, respec-
tively, from each dominant conformation determined by
the typical J values and ROE data. For the 1,3-methine
system of C4-C6, by analyzing the J values measured
at room temperature (Table 1), the conformation for C5-
C6 was easily determined, but it was difficult to assign
the conformation for C4-C5 because no dominant rota-
mer or a pair of alternating rotamers satisfied the J
values around the bond. However, when the spectra were
taken at 0 °C with the expectation of increasing the
population of a dominant conformer, the anti relationship
(10) Vining, L. C.; Taber, W. A. Can. J . Chem. 1962, 40, 1579-1584.
(11) (a) Otting, G.; Wuthrich, K. Quart. Rev. Biophys. 1990, 23, 39-
96. (b) Willborn, U.; Leibfritz, D. J . Magn. Reson. 1992, 98, 142-146.
(c) Kurz, M.; Schmieder, P.; Kessler, H. Angew. Ed. Engl. 1991, 30,
1329-1331.
(12) (a) Zhu, G.; Bax, A. J . Magn. Reson. 1993, 104A, 353-357. (b)
Zhu, G.; Live, D.; Bax, A. J . Am. Chem. Soc. 1994, 116, 8370-8371.
(c) Boyd, J .; Soffe, N.; J ohn, B.; Plant, D.; Hurd, R. J . Magn. Reson.
1992, 98, 660-664. (d) Davis, A. L.; Keeler, J .; Laue, E. D.; Moskau,
D. J . Magn. Reson. 1992, 98, 207-216.
(13) Rychnovsky, S. D.; Griesgraber, G.; Schlegel, R. J . Am. Chem.
Soc. 1995, 117, 197-210.

Absolute Configuration of Aflastatin A
J . Org. Chem., Vol. 65, No. 2, 2000 441
3
2
Ta ble 1. J H,H a n d J C,H Va lu es (Hz) a n d Key ROEs Used for Deter m in a tion of th e Con figu r a tion s fr om C4 to C8 a n d
fr om C23 to C31 of 7a
bond
coupled nuclei
3J H,H
coupled nuclei
2J C,H
ROE
C4-C5
H-4,H-5a
H-4,H-5b
H-5a,H-6
H-5b,H-6
H-6,H-7
7.5 (10.0b)
5.0
H-6,H-48b
C5-C6
C6-C7
4.5 (4.5b)
9.0 (9.0b)
4.0
H-5a,C6
H-5b,C6
H-6,C7
H-7,C6
-2.5
-5.5
-2.0
-1.2
C7-C8
C23-C24
H-7,H-8
8.0
3.0
8.0
3.0
8.0
7.5
3.0
H-6,H-49
H-23,H-24a
H-23,H-24b
H-24a,H-25
H-24b,H-25
H-25,H-26
H-26,H-27
H-24a,C23
H-24b,C23
H-24a,C25
H-24b,C25
0.0
-5.9
0.0
C24-C25
-5.0
C25-C26
C26-C27
H-24,H-27
H-26,C27
H-27,C26
H-27,C28
H-28,C27
H-28,C29
H-29,C28
H-30a,C29
H-30b,C29
H-30a,C31
H-30b,C31
1.0
0.0
C27-C28
C28-C29
C29-C30
C30-C31
H-27,H-28
H-28,H-29
5.0
3.0
-1.5
-2.0
0.0
0.0
H-29,H-30a
H-29,H-30b
H-30a,H-31
H-30b,H-31
8.0 (9.0b)
6.0 (5.0b)
-4.8 (-6.0b)
-5.0 (-3.0b)
-2.0
3.5 (3.5b)
10.0 (10.0b)
-6.0
a
b
Spectra were obtained in C5D5N at 27 °C or 0 °C. Measured at 0 °C.
between H-4 and H-5a was shown by the 3J H-4,H-5a value
(10 Hz). In addition to this J value, the observation of
ROE between H-6 and H-48 confirmed the conformation
for C4-C5 at 0 °C as shown in Figure 3b. Since the
action of 1; 1 inhibits the expression of mRNA of some
genes coding for aflatoxin biosynthetic enzymes.14 The
stereochemistry of 1 is very important for the study of
the interaction between 1 and its target molecule. Work
to investigate the molecular mechanism of inhibition of
aflatoxin production by 1 is now in progress.
3
3J H-6,H-5a and J H-6,H-5b values measured at 0 °C were
identical with those at room temperature, it was shown
that the conformation for C5-C6 was not changed by
lowering the temperature. Thus, the configuration for
C4-C6 was assigned as anti. Similarly, the conformation
for C29-C30 could not be deduced from the J values
measured at room temperature, but it could be assigned
by those obtained at 0 °C, as shown in Figure 3c. Since
the conformation for C30-C31 was clearly determined
Exp er im en ta l Section
Gen er a l Meth od s. NMR spectra were recorded at 27 °C
3
1
or 0 °C. J H,H values were extracted from 1D H NMR and 2D
2,3
E. COSY spectra.
J
values were obtained from phase-
C,H
sensitive (P)HETLOC,11b CH-HETLOC,11b and phase-sensi-
tive (P)FG-HMBC12c,d spectra, which were measured under the
following conditions. PHETLOC: BIRD delay, 275 ms or 500
3
by the J values, and both of the 3J H-30a,H-31 and J H-30b,H-31
2
3
values at 0 °C were the same as those at room temper-
ature, the configuration of C29-C31 was assigned as
shown in Figure 1.
ms; spin-lock period, 30 ms for J C,H or 60 ms for J C,H with
each 2.5 ms trim pulses; ∆, 3.45 ms. CH-HETLOC: BIRD
delay, 275, 350, or 500 ms; spin-lock period, 30 ms for J C,H or
2
3
60 ms for J C,H with each 1.0 ms trim pulses; ∆, 3.45 ms. PFG-
With respect to the C6-C7 diol system of 7, the
3
2
HMBC: delay, 40 ms or 50 ms; FG, grad1 (44), grad2 (4), grad3
(20), and grad4 (-20). Aflastatin A (1) was purified from the
mycelial methanol extracts of Streptomyces sp. MRI142 ac-
cording to the procedure previously reported.5a N-Methyl-D-
and N-methyl-L-alanine were purchased from Sigma, and (R)-
and (S)-isomers of 1,2,4-butanetriol were purchased from
Aldrich and Fluka, respectively.
F r a gm en ts 3 a n d 4. The degradation procedure to obtain
4 and the decaacetate of 3 and their spectral data have been
reported previously.5b,c Compound 3 was prepared from its
decaacetate as follows. To a solution of 3 decaacetate (28.5 mg)
in absolute MeOH (3.0 mL) was added a small piece of Na,
the mixture was stirred at room temperature for 40 min. After
the mixture was passed through a Dowex-50W (H+) column
(18 L × 40 mm), the solvent was removed under reduced
pressure to afford 3 (11.5 mg).
medium values of J H-6,H-7 (4.0 Hz), J H-6,C7 (-1.2 Hz),
2
and J C6,H-7 (-2.0 Hz) were obtained. Because these J
values are observed in the case of the pair of rotamers
(Figure 3d),7a in which H-6/7-OH and H-7/6-OH alter-
nated anti and gauche conformation, the configuration
of C6-C7 was assigned as threo. In the same manner,
the configuration for the C27-C28 diol system with the
3
2
J values of J H-27,H-28 (5.0 Hz), J H-27,C28 (-1.5 Hz), and
2J C27,H-28 (-2.0 Hz) was also assigned as threo (Figure
3d). Thus, all the configurations from C4 to C8 and from
C23 to C31 of 7 were determined. By connecting this
result with the configuration of 3, the relative stereo-
chemistry of 7 was assigned as shown in Figure 1,
affording the relative configurations from C6 to C33 of
1. These relative configurations were further connected
with the absolute configuration at C6 and C33 of 1 to
give their absolute configurations. Since the absolute
configurations from C8 to C31 of 1 obtained from the
configuration at C6 agreed with those based on that at
C33, the validity of the relative configurations from C6
to C33 of 1 determined by J -based method was confirmed.
From the results obtained above, the absolute config-
uration of aflastatin A was determined as 1. We have
recently obtained some information about the mode of
3: HRFABMS (positive, glycerol matrix) m/z 499.3481 (M
1
+ H)+ (calcd for C24H51O10, 499.3482); H NMR (CD3OD, 500
MHz) δ 4.02 (H-9), 4.01 (H-7), 3.96 (H-15), 3.95 (H-13), 3.93
(H-17), 3.83 (H-5), 3.76 (H-3), 3.69 (H-19a,b), 3.65 (H-11), 3.56
(H-1b), 3.47 (H-1a), 1.86 (H-2), 1.80 (H-10), 1.79 (H-4), 1.78
(H-6), 1.75 (H-8a), 1.73 (H-18a), 1.71 (H-14a), 1.68 (H-12), 1.63
(H-18b), 1.62 (H-14b), 1.62 (H-16a,b), 1.51 (H-8b), 0.95 (H-22),
0.93 (H-24), 0.87 (H-20), 0.81 (H-23), 0.78 (H-21); 13C NMR
(14) Sakurada, M.; Okamoto, S.; Ono, M.; Tsukigi, H.; Suzuki, A.;
Nagasawa, H.; Sakuda, S. Unpublished data.

442 J . Org. Chem., Vol. 65, No. 2, 2000
Ikeda et al.
(CD3OD, 125 MHz) δ 81.4 (C-5), 78.6 (C-11), 77.6 (C-3), 77.0
(C-7), 75.8 (C-13), 73.7 (C-9), 70.4 (C-15), 68.8 (C-17), 66.5 (C-
1), 60.0 (C-19), 45.4 (C-16), 43.1 (C-10), 42.6 (C-14), 41.0 (C-
18), 40.1 (C-12), 39.9 (C-6), 39.4 (C-4), 38.8 (C-2), 36.6 (C-8),
was washed with brine, sat NaHCO3 solution, and distilled
water and dried. After removal of the solvent, the resulting
residue was purified by normal-phase HPLC (mobile phase:
hexane-EtOAc, 100:1; column: Senshu pak Silica, 4.6 L ×
250 mm) to afford 9 (1.2 mg).
1
13.4 (C-21), 11.4 (C-23), 9.7 (C-20), 6.5 (C-22), 6.2 (C-24); H
NMR (C5D5N-CD3OD 3:1, 500 MHz) δ 4.33 (H-9), 4.28 (H-
15), 4.27 (H-7), 4.26 (H-17), 4.24 (H-13), 4.00 (H-3), 3.95 (H-
5), 3.94 (H-19b), 3.89 (H-19a), 3.86 (H-11), 3.82 (H-1b), 3.70
(H-1a), 2.05 (H-10), 1.95 (H-2), 1.90 (H-4,8a), 1.89 (H-14a), 1.88
(H-18a,b), 1.86 (H-16a), 1.85 (H-6), 1.81 (H-14b), 1.78 (H-8b),
1.78(H-16b), 1.77 (H-12), 1.06 (H-22), 1.04 (H-24), 0.97 (H-20),
0.84 (H-23), 0.69 (H-21); 13C NMR (C5D5N-CD3OD 3:1, 125
MHz) δ 80.9 (C-5), 78.2 (C-11), 76.7 (C-3), 76.4 (C-7), 75.4 (C-
13), 73.3 (C-9), 70.1 (C-15), 68.4 (C-17), 66.5 (C-1), 59.2 (C-
19), 44.9 (C-16), 42.2 (C-10), 42.2 (C-14), 40.7 (C-18), 39.2 (C-
12), 39.0 (C-6), 38.4 (C-4), 38.0 (C-2), 36.3 (C-8), 12.7 (C-21),
Natural-9: HRFABMS (positive, NBA matrix) m/z 355.1898
(M + H)+ (calcd for C22H27O4, 355.1909); 1H NMR (CDCl3, 500
MHz) δ 4.36 (H-6a,b), 4.21 (H-1a, dd, J ) 5.5, 11 Hz), 4.09
(H-1b, dd, J ) 6.5, 11 Hz), 2.07 (H-2), 1.84 (H-4, 5a), 1.55 (H-
5b), 1.50 (H-3a), 1.16 (H-3b), 1.03 (H-7, CH3, d, J ) 6.5 Hz),
1.01 (H-8, CH3, d, J ) 6.5 Hz), 8.00 (Bz-meta), 7.52 (Bz-para),
7.40 (Bz-ortho); 13C NMR (CDCl3, 125 MHz) δ 69.6 (C-1), 63.2
(C-6), 41.2 (C-3), 35.3 (C-5), 30.1 (C-2), 27.3 (C-4), 20.1 (C-8),
17.7 (C-7), 166.7 (Bz-ester), 132.8 (Bz-para), 130.4 (Bz), 129.5
(Bz-meta), 128.3 (Bz-ortho); CD (CH3CN) ∆ꢀ230 ) +1.52; [R]26
D
+8.5° (c 0.1, CHCl3).
11.0 (C-23), 9.3 (C-20), 5.9 (C-22), 5.6 (C-24); [R]23 -4.03° (c
(2R,4R)- a n d (2S,4R)-2,4-Dim eth ylcycloh exa n on e (11).
(2R,4R)- and (2S,4R)-11 were prepared according to the
literature procedure.9 In brief, a solution of 10 (5.0 g) in 10%
NaOH solution (50 mL) was stirred at -4 °C for 8 h and then
at -18 °C for 14 h. The petroleum ether extract of the reaction
mixture was purified by normal-phase HPLC (mobile phase:
hexane-EtOAc, 100:1; column: SSC-silica 4251-N, 10 L × 250
mm) to afford (2R,4R)-11 (730 mg) and (2S,4R)-11 (69.5 mg)
as a colorless oil. (2R, 4R)-(11): FABMS (positive, NBA matrix)
D
0.6, EtOH).
3-Hyd r oxyd od eca n oic Acid (5). A solution of 1 (80 mg)
in MeOH (20 mL) was added to 0.5 M NaIO4 aq solution (6.4
mL), and the mixture was stirred for 20 h at room tempera-
ture. After being quenched with ethylene glycol (0.24 mL), the
reaction mixture was filtered and the solvent was removed
under reduced pressure. The product was purified by reverse-
phase HPLC (mobile phase: 50% CH3CN in H2O; column:
PEGASIL ODS, 10 L × 250 mm) to afford 5 (1.4 mg).
5: HRFABMS (positive, NBA matrix) m/z 239.1617 (M +
Na)+ (calcd for C12H24O3Na, 239.1623); 1H NMR (CD3OD, 500
MHz) δ 4.36 (H-6a,b), 3.96 (H-3), 2.43 (H-2a, dd, J ) 4.5, 15
Hz), 2.36 (H-2b, dd, J ) 8.5, 15 Hz), 1.46 (H-4 and H-5a), 1.30
(H-5b, H-6, H-7, H-8, H-9, H-10 and H-11), 0.89 (H-12, t, J )
7.0 Hz); 13C NMR (CDCl3, 125 MHz) δ 175.8 (C-1), 69.4 (C-3),
43.3 (C-2), 38.1 (C-4), 33.1 (C-10), 30.7, 30.5, and 26.7 (C-5,
1
m/z 149 (M + Na)+; H NMR (CDCl3, 500 MHz) δ 2.35 (H-2),
2.27 (H-6), 1.98 (H-5eq), 1.95 (H-3eq), 1.89 (H-4), 1.28 (H-5ax),
1.05 (H-3ax), 0.91 (H-7, CH3, d, J ) 6.5 Hz), 0.90 (H-8, CH3,
d, J ) 6.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 213.7 (C-1), 44.5
(C-3), 44.3 (C-2), 41.2 (C-6), 35.9 (C-5), 32.0 (C-4), 21.2 (C-8),
14.5 (C-7); [R]23 -7.6° (c 1.7, CHCl3).
D
(2S,4R)-(11): FABMS (positive, NBA matrix) m/z 149 (M +
Na)+; 1H NMR (CDCl3, 500 MHz) δ 2.52 (H-2a, b), 2.34 (H-
3eq, 6eq), 2.06 (H-4), 1.90 (H-5eq), 1.70 (H-3ax), 1.65 (H-6ax),
1.58 (H-5ax), 1.08 (H-7, CH3, d, J ) 6.5 Hz), 1.07 (H-8, CH3,
d, J ) 6.5 Hz); 13C NMR (CDCl3, 125 MHz) δ 215.0 (C-1), 41.8
(C-2), 41.5 (C-3), 37.4 (C-6), 33.9 (C-5), 26.5 (C-4), 19.4 (C-7),
C-6, C-7, C-8 and C-9), 23.7 (C-11), 14.4 (C-12); [R]28 -16.0°
D
(c 0.1, CHCl3) [lit.10 [R]25 -15.2° (c 1.6, CHCl3)].
D
Na tu r a l N-Meth yla la n in e a n d P r oced u r e of Ma r fey’s
Meth od . A solution of 4 (1.0 mg) in MeOH (0.5 mL) was added
to 0.04 M NaIO4 aq solution (1.5 mL), and the mixture was
stirred for 16 h at room temperature. After being quenched
with ethylene glycol (80 µL), the reaction solution was con-
centrated in vacuo. The obtained residue was dissolved in 6
N HCl (3.0 mL), and the solution was refluxed for 6 h. After
the reaction solution was concentrated to dryness, the residue
was dissolved in distilled water (1.5 mL). Ten microliters of
this solution was mixed with 1 M NaHCO3 solution (40 µL), a
solution of 1% 1-fluoro-2,4-dinitrophenyl-5-L-alanineamide
(FDAA) in acetone (40 µL), and distilled water (910 µL), and
the reaction mixture was maintained at 40 °C for 1 h. After
cooling, this solution (50 µL) was subjected to reverse-phase
HPLC (column: SSC-ODS-1151-N, 4.6 L × 150 mm; flow
rate: 1 mL/min; mobile phase: gradient elution of 10-50%
CH3CN in 10 mM AcONa buffer pH 5.5 in 35 min). The
retention times of FDAA derivatives of N-methyl-L-alanine and
N-methyl-D-alanine, and natural sample, were 19.0, 20.4, and
20.4 min, respectively.
16.0 (C-8); [R]23 +64.2° (c 0.44, CHCl3).
D
(2S,4S)-2,4-Dim eth yl-1,6-h exa n ed iol Diben zoa te (9). A
mixture of (2S,4R)-11 (61.6 mg) and Pb(OAc)4 (160 mg) in
benzene (1.0 mL) was heated under reflux for 8 h. After Et2O
(150 mL) was added to the reaction mixture, the organic
solution was washed with brine and distilled water, dried, and
evaporated. The resulting residue was chromatographed on a
silica gel column (hexane-EtOAc, 9:1). Crude acetoxylated
product (32.9 mg) that eluted from the column was dissolved
in CH2Cl2 (1.3 mL), and NaH2PO4 (140 mg) and mCPBA (90
mg) were added to the solution. The mixture was stirred at
room temperature for 24 h. After CH2Cl2 (50 mL) was added
to the reaction mixture, the organic solution was washed with
brine, sat NaHCO3 solution, and distilled water, dried, and
evaporated. Without purifying the lactone (2S,4S)-12, the
resulting crude product (82.3 mg) was treated with LiAlH4 (31
mg) in Et2O (5.0 mL) at room temperature for 2 h. After cooled
distilled water (10 mL) and 3 N HCl (4.0 mL) were added to
the reaction solution, the product was extracted with Et2O (50
mL). The ether layer was washed with distilled water, dried,
and evaporated. The resulting residue (228 mg) was dissolved
in dry pyridine (7.0 mL), and BzCl (3.5 mL) was added to the
solution. After being stirred at room temperature for 24 h, the
reaction was stopped by adding distilled water (5.0 mL). The
product was extracted with CH2Cl2 (50 mL), and the organic
layer was washed with sat NaHCO3 solution, brine, and
distilled water, dried, and evaporated. The resulting residue
was purified by silica gel column chromatography (hexane-
EtOAc, 7:3) and normal-phase HPLC (mobile phase: hexane-
EtOAc, 100:1; column: SSC-silica 4251-N, 10 L × 250 mm) to
afford (2S,4S)-9 (13.6 mg, 7.9%) as a colorless oil.
Na tu r a l 2,4-Dim eth yl-1,6-h exa n ed iol Diben zoa te (9).
Ozone was passed through a solution of 4 (7.9 mg) in absolute
MeOH (1.0 mL) at -78 °C for 30 min. After removal of excess
O3 in the solution by passage of N2, the reaction flask was
allowed to reach room temperature. Dimethyl sulfide (1.0 mL)
was added to the solution, and the resulting mixture was kept
at room temperature for 30 min. The product was extracted
with EtOAc, and the EtOAc solution was washed with brine
and distilled water and dried. After removal of the solvent,
the resulting residue was treated with LiAlH4 (5.0 mg) in Et2O
(2.0 mL) at room temperature for 1.5 h. After adding distilled
water (10 mL) and 3 N HCl (3.0 mL) to the reaction solution,
the product was extracted with Et2O. The ether layer was
washed with distilled water, dried, and evaporated. The
resulting residue was dissolved in dry CH3CN (1.0 mL). Tri-
n-butylamine (20 mL) and benzoyl cyanide (6.2 mg) were added
to the solution, and the mixture was stirred at room temper-
ature for 2 h. The reaction was stopped by adding distilled
water (10 mL), and the solution was stirred for 30 min. The
product was extracted with CH2Cl2, and the CH2Cl2 solution
(2S,4S)-(9): HRFABMS (positive, NBA matrix) m/z 355.1900
(M + H)+ (calcd for C22H27O4, 355.1909); 1H NMR (CDCl3, 500
MHz) δ 4.36 (H-6a, b), 4.18 (H-1a, dd, J ) 6.5, 11 Hz), 4.10
(H-1b, dd, J ) 6.5, 11 Hz), 2.05 (H-2), 1.80 (H-4, 5a), 1.64 (H-
5b), 1.31 (H-3a, b), 1.00 (H-7, CH3, d, J ) 7.0 Hz), 0.96 (H-8,
CH3, d, J ) 6.5 Hz), 8.02 (Bz-meta), 7.53 (Bz-para), 7.41 (Bz-
ortho); 13C NMR (CDCl3, 125 MHz) δ 70.2 (C-1), 63.3 (C-6),

Absolute Configuration of Aflastatin A
J . Org. Chem., Vol. 65, No. 2, 2000 443
40.8 (C-3), 36.3 (C-5), 30.2 (C-2), 27.3 (C-4), 19.3 (C-8), 16.7
(C-7), 166.6 (Bz-ester), 132.8 (Bz-para), 130.4 (Bz), 130.2 (Bz),
129.5 (Bz-meta), 128.3 (Bz-ortho); CD (CH3CN) ∆ꢀ237 ) -1.39;
from (R)- and (S)-1,2,4-butanetriol, respectively, by using the
same benzoylation method as that used to prepare natural-6.
(R)-6: HRFABMS (positive, NBA matrix) m/z 419.1501 (M
[R]23 -9.7° (c 0.18, CHCl3).
+ H)+ (calcd for C25H23O6, 419.1495); CD (CH3CN) ∆ꢀ230
)
D
(2R,4S)-2,4-Dim eth yl-1,6-h exa n ed iol Diben zoa te (9).
Crude lactone (2R,4S)-12 was prepared from (2R,4R)-11 in the
same manner as crude (2S,4S)-12 was prepared from (2S,4R)-
11. When this crude (2R,4S)-12 was used for the next LiAlH4
reduction as in the case of the preparation of (2S,4S)-9, pure
(2R,4S)-12 was not obtained by the final HPLC because of
contamination of byproducts. Thus, crude (2R,4S)-12 (153.5
mg) was purified by normal-phase HPLC (mobile phase:
hexane-2-PrOH, 150:1; column: SSC-silica 4251-N, 10 L ×
250 mm) to afford (2R,4S)-12 (56.7 mg) before the reduction.
(2R,4S)-12: FABMS (positive, NBA matrix) m/z 201 (M +
+4.8; [R]18 +45.3° (c 2.3, CHCl3).
D
(S)-6: HRFABMS (positive, NBA matrix) m/z 419.1477 (M
+ H)+ (calcd for C25H23O6, 419.1495); CD (CH3CN) ∆ꢀ230
)
-3.8; [R]18 -50.9° (c 1.2, CHCl3).
D
P en ta a ceton id e of F r a gm en t 3 (13). 2,2-Dimethoxypro-
pane (0.5 mL) and camphorsulfonic acid (25 mg) were added
to a solution of 3 (8.4 mg) in dry acetone (1.5 mL), and the
solution was stirred at room temperature for 24 h. After adding
triethylamine (0.3 mL), the reaction solution was concentrated
under reduced presssure. The resulting residue was purified
by silica gel column chromatography (hexane-EtOAc, 8:2) and
normal-phase HPLC (mobile phase: hexane-2-PrOH, 400:1;
column: PEGASIL silica, 4.6 L × 250 mm) to afford 13 (0.8
mg).
1
H)+, H NMR (CDCl3, 500 MHz) δ 6.45 (H-6), 2.63 (H-2), 2.01
(H-5a), 1.96 (H-4), 1.71 (H-5b), 1.66 (H-3a), 1.33 (H-3b), 1.19
(H-7, CH3), 0.99 (H-8, CH3), 2.10 (Ac CH3).
Compound (2R,4S)-12 was converted to (2R,4S)-9 by LiAlH4
reduction and benzoylation using the same method as that
used to convert crude (2S,4S)-12 to (2S,4S)-9. Final HPLC
purification (mobile phase: hexane-EtOAc, 100:1; column:
SSC-silica 4251-N, 10 L × 250 mm) afforded (2R,4S)-9 (13.5
mg) as a colorless oil.
13: HRFABMS (positive, NBA matrix) m/z 721.4835 (M +
Na)+ (calcd for C39H70O10Na, 721.4867); 1H NMR (benzene-d6,
500 MHz) δ 4.49 (H-7), 4.17 (H-3, dd, J ) 2.0, 7.0 Hz), 4.13
(H-17, dd, J ) 2.5, 6.5 Hz), 4.09 (H-15), 3.98 (H-5, dd, J ) 2.0,
9.0 Hz), 3.95 (H-13), 3.93 (H-1a), 3.69 (H-19b), 3.68 (H-19a),
3.64 (H-11, dd, J ) 2.0, 8.0 Hz), 3.53 (H-9), 3.46 (H-1b, dd, J
) 1.5, 6.5 Hz), 2.30 (H-4), 2.14 (H-16a), 1.90 (H-8a), 1.84 (H-
12), 1.80 (H-10), 1.77 (H-8b), 1.70 (H-14a), 1.63 (H-16b), 1.55
(H-6), 1.55 (H-18a), 1.55 (H-27, CH3), 1.54 (H-30, CH3), 1.53
(H-39, CH3), 1.50 (H-36, CH3), 1.49 (H-33, CH3), 1.47 (H-14b),
1.39 (H-28, CH3), 1.36 (H-25, CH3), 1.36 (H-31, CH3), 1.34 (H-
2), 1.33 (H-18b), 1.33 (H-37, CH3), 1.30 (H-34, CH3), 1.24 (H-
24, CH3, d, J ) 6.5 Hz), 1.16 (H-20, CH3, d, J ) 7.0 Hz), 1.03
(H-22, CH3), 0.95 (H-21, CH3, d, J ) 7.5 Hz), 0.61 (H-23, CH3,
d, J ) 6.5 Hz); 13C NMR (benzene-d6, 125 MHz) δ 74.3 (C-5),
73.1 (C-11), 72.0 (C-9), 71.6 (C-3), 71.6 (C-13), 70.2 (C-7), 68.3
(C-1), 65.5 (C-15), 65.5 (C-17), 59.8 (C-19), 44.1 (C-16), 39.8
(C-12), 38.3 (C-4), 36.4 (C-8), 35.0 (C-10), 35.0 (C-14), 32.8 (C-
6), 31.5 (C-18), 30.5 (C-2), 13.0 (C-20), 11.6 (C-23), 10.1 (C-
(2R,4S)-(9): HRFABMS (positive, NBA matrix) m/z 355.1896
(M + H)+ (calcd for C22H27O4, 355.1909); 1H NMR (CDCl3, 500
MHz) δ 4.36 (H-6a, b), 4.21 (H-1a, dd, J ) 5.5, 11 Hz), 4.09
(H-1b, dd, J ) 6.5, 11 Hz), 2.07 (H-2), 1.84 (H-4, 5a), 1.55 (H-
5b), 1.50 (H-3a), 1.16 (H-3b), 1.03 (H-7, CH3, d, J ) 6.5 Hz),
1.01 (H-8, CH3, d, J ) 6.5 Hz), 8.00 (Bz-meta), 7.52 (Bz-para),
7.40 (Bz-ortho); 13C NMR (CDCl3, 125 MHz) δ 69.6 (C-1), 63.2
(C-6), 41.2 (C-3), 35.3 (C-5), 30.1 (C-2), 27.3 (C-4), 20.1 (C-8),
17.7 (C-7), 166.6 (Bz-ester), 132.8 (Bz-para), 130.4 (Bz), 129.5
(Bz-meta), 128.3 (Bz-ortho); CD (CH3CN) ∆ꢀ230 ) -1.96; [R]26
-8.9° (c 0.57, CHCl3).
D
Na tu r a l 1,2,4-Bu ta n etr iol Tr iben zoa te (6). A solution of
1 (80 mg) in 5% HCl-MeOH (1.0 mL) was stirred at room
temperature for 2 h. The reaction mixture was purified by
reverse-phase HPLC (mobile phase: 63% MeOH in 0.5%
diethylamine; column: Capcell pak C18, 10 φ × 250 mm) to
afford 2 (33 mg). 2: FABMS (positive, glycerol matrix) m/z
1316 (M + 2Na - H)+.
21), 9.6 (C-24), 6.1 (C-22), acetonide moieties (Figure 4); [R]23
D
-0.4° (c 0.1, CHCl3).
F r a gm en t 7. Ozone was passed through a solution of the
2 (40 mg) in absolute MeOH at -78 °C for 20 min. After
removal of excess O3 by passage of N2, NaBH4 (35 mg) was
added to the solution and the reaction flask was allowed to
reach room temperature. The reaction mixture was stirred at
room temperature for 2 h, and the reaction was stopped by
adding acetic acid (0.2 mL). After removal of solvent and boron,
a mixture of dry pyridine (6.0 mL), acetic anhydride (3.0 mL),
and 4-(dimethylamino)pyridine (3.0 mg) was added to the
residue. The reaction mixture was stirred at room temperature
for 22 h, and the reaction was stopped by adding cooled
distilled water (3.0 mL). The solution was extracted with
EtOAc, and the EtOAc layer was washed with brine, sat
NaHCO3 solution, and distilled water and dried. After evapo-
ration in vacuo, the resulting residue was purified by reverse-
phase HPLC (mobile phase: 70% MeOH in H2O; column:
Capcell pak C18, 10 L × 250 mm) to afford peracetate of 7 (34.0
mg). To a solution of 7 peracetate (34.0 mg) in absolute MeOH
(3.0 mL), a small piece of Na was added and stirred at room
temperature for 40 min. After being passed through a Dowex-
50W (H+) column (18 L × 40 mm), the solvent was removed
under reduced pressure to afford 7 (19.7 mg).
A mixture of 2 (16 mg) in MeOH (4.0 mL) and 0.2 M NaIO4
aq solution (1.0 mL) was stirred at room temperature for 3 h,
and the reaction was stopped by adding ethylene glycol (0.1
mL). The reaction mixture was filtered with a glass filter, and
the solvent was removed under reduced pressure. The result-
ing residue was dissolved in MeOH (1.0 mL), and NaBH4 (26
mg) was added to the solution. The reaction mixture was
stirred at room temperature for 2.5 h, and the reaction was
quenched by addition of acetic acid (120 µL). After removal of
the solvent and boron, the resulting residue was dissolved in
3 N HCl (3.0 mL). The solution was heated at 80 °C for 3.5 h
and evaporated. The residue was dissolved in dry pyridine (4.0
mL), and 4-(dimethylamino)pyridine (2.0 mg) and benzoyl
chloride (1.5 mL) were added to the solution. After being
stirred at room temperature for 24 h, the reaction was stopped
by adding cooled distilled water (3.0 mL). The product was
extracted with EtOAc, and the EtOAc solution was washed
with brine, sat NaHCO3 solution, and distilled water, dried,
and evaporated. The obtained residue was purified by reverse-
phase HPLC (mobile phase: gradient elution of 20-90% CH3-
CN in H2O; column: Capcell pak C18, 10 L × 250 mm) to afford
6 (1.3 mg).
7: HRFABMS (positive, glycerol matrix) m/z 1117.7025 (M
+ Na) + (calcd for C53H106O22Na, 1117.7073); 1H NMR (C5D5N,
600 MHz) δ 4.96 (H-27), 4.92 (H-29), 4.67 (H-23 and H-26),
4.64 (H-34), 4.61 (H-15), 4.56 (H-33), 4.49 (H-13, H-21and
H-28), 4.42 (H-19), 4.35 (H-25), 4.32 (H-9 and H-32), 4.29 (H-
6), 4.24 (H-31), 4.18 (H-37), 4.12 (H-11), 4.05 (H-17), 4.00 (H-
7), 3.80 (H-1a), 3.61 (H-1b), 3.17 (H-30a), 2.59 (H-24a), 2.49
(H-30b), 2.45 (H-36a), 2.31 (H-8), 2.19 (H-16 and H-36b), 2.14
(H-4 and H-24b), 2.08 (H-10), 2.05 (H-22a), 2.04 (H-14a and
H-20a), 1.98 (H-2 and H-22b), 1.97 (H-12), 1.95 (H-14b), 1.86
(H-18 and H-20b), 1.84 (H-5a), 1.78 (H-3a), 1.75 (H-5b), 1.67
(H-38a), 1.59 (H-38b and H-39a), 1.48 (H-39b), 1.28 (H-49),
1.25 (H-40a,b), 1.23 (H-45a,b and H-51), 1.19 (H-41,H-42,H-
43,H-44 and H-53), 1.10 (H-47), 1.07 (H-48), 1.04 (H-3b), 0.97
Natural-6: HRFABMS (positive, NBA matrix) m/z 419.1491
(M + H)+ (calcd for C25H23O6, 419.1495); 1H NMR (CDCl3, 500
MHz) δ 5.72 (H-2), 4.65 (H-1a, dd, J ) 3.5, 12 Hz), 4.56 (H-
4a), 4.53 (H-1b), 4.45 (H-4b), 2.34 (H-3), 8.03-7.98 (Bz-meta),
7.55-7.49 (Bz-para), 7.41-7.36 (Bz-ortho); 13C NMR (CDCl3,
125 MHz) δ 69.5 (C-2), 65.5 (C-1), 61.0 (C-4), 30.4 (C-3), 166.4,
166.2, 165.9 (Bz ester), 133.2, 133.0 (Bz-meta), 129.7, 129.6
(Bz-para), 128.4 (Bz-ortho); CD (CH3CN) ∆ꢀ230 ) +3.8; [R]19
D
+63.0° (c 0.1, CHCl3).
(R)- a n d (S)-1,2,4-Bu ta n etr iol Tr iben zoa te (6). Authen-
tic (R)- and (S)-1,2,4-butanetriol tribenzoate were prepared

444 J . Org. Chem., Vol. 65, No. 2, 2000
Ikeda et al.
(H-52), 0.83 (H-46), 0.81 (H-50); 13C NMR (C5D5N, 150 MHz)
δ 103.2 (C-35), 81.6 (C-11), 78.8 (C-9), 78.8 (C-17), 77.9 (C-7),
77.1 (C-13), 76.2 (C-19), 76.2 (C-25), 75.4 (C-28), 73.9 (C-15),
73.1 (C-23), 72.8 (C-31), 72.6 (C-33), 72.3 (C-32), 72.2 (C-26),
71.5 (C-27), 71.5 (C-34), 70.9 (C-29), 70.8 (C-21), 69.5 (C-6),
67.2 (C-1), 66.9 (C-37), 45.7 (C-14), 42.7 (C-16), 42.7 (C-20),
42.3 (C-5), 42.1 (C-24), 41.6 (C-3), 39.5 (C-18), 39.3 (C-12), 39.3
(C-36), 39.3 (C-38), 38.8 (C-10), 38.1 (C-8), 37.5 (C-30), 36.9
(C-22), 34.0 (C-2), 32.0 (C-44), 30.0 (C-41), 30.0 (C-42), 29.8
(C-40), 29.5 (C-43), 27.8 (C-4), 26.0 (C-39), 22.9 (C-45), 21.7
(C-48), 18.6 (C-47), 14.2 (C-46), 13.2 (C-50), 11.6 (C-52), 8.3
(C-49), 6.4 (C-51), 6.1 (C-53).
of Tokyo for helpful discussions. This work was sup-
ported by a grant from J SPS Program for Research for
the Future.
Su p p or t in g In for m a t ion Ava ila b le: 13C and 1H NMR
1
spectra of 3, 5-7, 9, 11, and 13, the H NMR spectrum of 12,
COSY, FG-HMQC, FG-HMBC, NOESY spectra and summary
of NMR data of 13, and partial HETLOC, sliced PS-HMBC
and ROESY spectra of 3 and 7 used for their configuration
assignments. This material is available free of charge via the
Internet at http://pubs.acs.org.
Ack n ow led gm en t. We thank Dr. M. Murata of
Osaka University and Dr. K. Furihata of the University
J O991284C